Methods for corn transformation

ABSTRACT

The present invention relates to a novel transformation system for generating transformed corn plants. In particular, the invention relates to a rapid selection system at an elevated temperature that allows faster and more efficient transformation.

[0001] This application claims priority to U.S. Provisional Application60/474,589, filed May 30, 2004, herein incorporated by reference in itsentirety.

BACKGROUND OF INVENTION

[0002] The present invention relates to the field of plantbiotechnology. More specifically, it concerns methods for incorporatinggenetic components into a plant via genetic engineering techniques. Inparticular, provided herein are systems for genetically transformingcorn.

[0003] During the past decade, it has become possible to transfer genesfrom a wide range of organisms to crop plants by recombinant DNAtechnology. This advance has provided enormous opportunities to improveplant resistance to pests, disease and herbicides, and to modifybiosynthetic processes to change the quality of plant products for food,feed and industrial uses.

[0004]Agrobacterium-mediated transformation is one method forintroducing a desired genetic element into a plant and is achievedthrough the use of a genetically engineered soil bacterium belonging tothe genus Agrobacterium. Several Agrobacterium species mediate thetransfer of a specific DNA known as “T-DNA” that can be geneticallyengineered to carry a desired piece of DNA into many plant species. Themajor events marking the process of T-DNA mediated pathogenesis areinduction of virulence genes, processing and transfer of T-DNA.

[0005]Agrobacterium-mediated genetic transformation of plants involvesseveral steps. The first step, in which the virulent Agrobacterium andplant cells are first brought into contact with each other, is generallycalled “inoculation”. Following the inoculation step, the Agrobacteriumand plant cells/tissues are usually grown together for a period ofseveral hours to several days or more under conditions suitable forgrowth and T-DNA transfer. This step is termed “co-culture”. Followingco-culture and T-DNA delivery, the plant cells are often treated withbactericidal or bacteriostatic agents to kill the Agrobacterium. If thisis done in the absence of any selective agents to promote preferentialgrowth of transgenic versus non-transgenic plant cells, then this istypically referred to as the “delay” step. If done in the presence ofselective pressure favoring transgenic plant cells, then it is referredto as a “selection” step. When a “delay” is used, it is typicallyfollowed by one or more “selection” steps. Both the “delay” and“selection” steps typically include bactericidal or bacteriostaticagents to kill any remaining Agrobacterium cells because the growth ofAgrobacterium cells is undesirable after the infection (inoculation andco-culture) process.

[0006] Another widely used technique to genetically transform plantsinvolves the use of microprojectile bombardment. In this process, anucleic acid containing the desired genetic elements to be introducedinto the plant is deposited on small metallic, e.g., gold or tungsten,particles, which are then delivered at a high velocity into the planttissue or plant cells. Cells containing the desired genetic elements arethen placed in tissue culture and transformed cells selected through theuse of one or more selection system that has been incorporated into thegenetic elements transformed into the plant.

[0007] The major deficiencies in current plant transformation systemsinclude but are not limited to the production efficiency of the system,and transformation variability due to genotype or species diversity andexplant limitations. In particular, there is a continuing need in thefield of plant biotechnology to provide more efficient transformationmethods suitable for high capacity production of economically importantplants, particularly elite cultivars.

SUMMARY OF INVENTION

[0008] The present invention provides novel methods for the stable andefficient transformation of cereal plants, particularly corn (maize orZea mays) plants, under selection conditions that enable a rapid andefficient transformation process.

[0009] In one aspect, the present invention provides a method oftransforming corn plants by introducing into transformable corn tissuevia a biotechnological transformation process a nucleic acid sequencecontaining at least one genetic component containing a selectable markernucleic acid that provides a means for selecting corn tissue containingthe genetic component from those not containing the genetic component,and subjecting the transformed corn tissue to tissue culture mediacontaining a selection agent corresponding to the selectable markernucleic acid at an elevated temperature for a period of time sufficientto identify and select transformed plants (or tissue) containing thedesired genetic component.

[0010] Still another aspect of the present invention relates to a methodfor transforming corn tissue with a genetic component containing atleast one selectable marker nucleic acid and subjecting the transformedtissue to a tissue culture media containing the corresponding selectionagent for a period of not more than twenty-one days and identifying andselecting transformed corn tissue containing the desired geneticcomponent for regeneration into a fertile, transgenic plant.

[0011] In a still further aspect of the present invention, the selectionstep is conducted in a single tissue culture media vessel containing theselective agent, and the tissue surviving selection is immediatelytransferred to a regeneration media for development into fertile,transgenic maize plants.

[0012] In another aspect of the present invention, the transformablecorn tissue is transformed via Agrobacterium-mediated transformationmethods wherein the exposure of the corn tissue to the Agrobacterium isminimized, it is inoculated by methods that limit its exposure toanerobiosis conditions during the Agrobacterium inoculation. Variousmeans for minimizing exposure may be utilized including limiting thetime of submersion in the Agrobacterium solution, inoculating with asmall drop of Agrobacterium solution, and inoculating with filter papersaturated with the Agrobacterium solution.

[0013] Yet another aspect of the present invention relates to any seeds,or progeny including hybrid combinations of the transformed plantsproduced by the methods of the present invention.

[0014] Further objects, advantages and aspects of the present inventionwill become apparent from the accompanying figures and followingdescription of the invention.

BRIEF DESCRIPTION OF DRAWINGS

[0015]FIG. 1 is a plasmid map of pMON30113.

[0016]FIG. 2 is a plasmid map of pMON42071.

[0017]FIG. 3 is a plasmid map of pMON65324.

DETAILED DESCRIPTION

[0018] The present invention is particularly directed to thetransformation of any cereal plant, particularly corn lines. The presentinvention provides for a rapid and efficient process for obtainingtransformed corn plants by reducing the time the transformable explantsspend in the selection phase, which in this system is the callusinduction phase, of a typical transformation process and by increasingthe temperature during the selection phase. It has been discovered thatplacing the transformed corn explant on a tissue culture mediacontaining a selective agent for a total period of 2-4 weeks issufficient to select for transformed plants and to obtain fertiletransgenic plants therefrom. Moreover, the transformed explants may beplaced in a single vessel containing the selection media for theentirety of the selection period with efficacious results, thuseliminating the need for repeated subculturing and transfer of theexplants to fresh media. In this method, it is often desirable tomaintain selection pressure during the regeneration phase as well.

[0019] It has further been discovered that the transformation efficiencymay be increased about twofold by maintaining the temperature during theselection phase, or at least a portion of the selection phase, at atemperature greater than 27° C., a typical corn tissue culturetemperature. Preferably, the selection temperature is maintained betweenabout 28.5° C. and about 35° C., more preferably the temperature ismaintained between about 30° C. and about 34° C., and most preferablythe temperature is maintained between about 30° C. and about 32° C. Thiselevated temperature is maintained for a period of not more than threeweeks and more preferably not more than two weeks during the selectionphase.

[0020] It has also been discovered that limiting the amount ofsubmersion during Agrobacterium inoculation of the transformableexplants increases the transformation frequency. The explants can beisolated directly into inoculation medium and then removed from themedium within 20 minutes. Explants could also be isolated into coculturemedium and then spotted with 1 μL of Agrobacterium solution or contactedwith filter paper saturated with Agrobacterium solution for 5 to 60minutes. It is believed that the anaerobiosis of being in solution isdetrimental to the explants. Thus limiting or eliminating theanaerobiosis increases the transformation efficiency. It has beenobserved that immersion in the inoculation medium for more than 60minutes has a detrimental effect on the explant. Thus eliminating theanaerobiosis by altering the manner or the time in which theAgrobacterium contacts the explant increases the transformationefficiency.

[0021] Cereal plants include monocotyledonous plants such as wheat,rice, barley, oats, sorghum, and maize.

[0022] Transformation efficiency or transformation frequency is thepercentage of transformants produced from the starting explants. Thepresent invention is directed to methods that increase the number oftransformants produced from a given number of explants.

[0023] The present invention involves the delivery of a desired nucleicacid, a gene of interest, into the genome of a corn cell and theproduction of a fertile transgenic plant therefrom. The method ofdelivery of the nucleic acid is not critical to the methods of thisinvention and known delivery methods such as Agrobacterium-mediateddelivery and microprojectile delivery methods may be used. Thus, in oneaspect, the present invention encompasses the use of bacterial strainsto introduce one or more genetic components into plants. Those of skillin the art would recognize the utility of Agrobacterium-mediatedtransformation methods. A number of wild-type and disarmed strains ofAgrobacterium tumefaciens and Agrobacterium rhizogenes harboring Ti orRi plasmids can be used for gene transfer into plants. Preferably, theAgrobacterium hosts contain disarmed Ti and Ri plasmids that do notcontain the oncogenes that cause tumorigenesis or rhizogenesis,respectfully, which are used as the vectors and contain the genes ofinterest that are subsequently introduced into plants. Preferred strainswould include but are not limited to Agrobacterium tumefaciens strainC58, a nopaline-type strain that is used to mediate the transfer of DNAinto a plant cell, octopine-type strains such as LBA4404orsuccinamopine-type strains, e.g., EHA101 or EHA105. The use of thesestrains for plant transformation has been reported and the methods arefamiliar to those of skill in the art.

[0024] With respect to microprojectile bombardment (U.S. Pat. Nos.5,550,318; U.S. Pat. No. 5,538,880; U.S. Pat. No. 5,610,042; each ofwhich is specifically incorporated herein by reference in its entirety),particles are coated with nucleic acids and delivered into cells by apropelling force. Exemplary particles include those comprised oftungsten, platinum, and preferably, gold. It is contemplated that insome instances DNA precipitation onto metal particles would not benecessary for DNA delivery to a recipient cell using microprojectilebombardment. However, it is contemplated that particles may contain DNArather than be coated with DNA. Hence, it is proposed that DNA-coatedparticles may increase the level of DNA delivery via particlebombardment but are not, in and of themselves, necessary.

[0025] For the bombardment, cells in suspension are concentrated onfilters or solid culture medium. Alternatively, immature embryos orother target cells may be arranged on solid culture medium. The cells tobe bombarded are positioned at an appropriate distance below themicroprojectile stopping plate.

[0026] An illustrative embodiment of a method for delivering DNA intoplant cells by acceleration is the Biolistics Particle Delivery System(BioRad, Hercules, Calif.), which can be used to propel particles coatedwith DNA or cells through a screen, such as a stainless steel or Nytexscreen, onto a filter surface covered with monocot plant cells culturedin suspension. The screen disperses the particles so that they are notdelivered to the recipient cells in large aggregates. It is believedthat a screen intervening between the projectile apparatus and the cellsto be bombarded reduces the size of projectiles aggregate and maycontribute to a higher frequency of transformation by reducing thedamage inflicted on the recipient cells by projectiles that are toolarge.

[0027] For microprojectile bombardment, one will attach (i.e., “coat”)DNA to the microprojectiles such that it is delivered to recipient cellsin a form suitable for transformation thereof. In this respect, at leastsome of the transforming DNA must be available to the target cell fortransformation to occur, while at the same time during delivery the DNAmust be attached to the microprojectile. Therefore, availability of thetransforming DNA from the microprojectile may comprise the physicalreversal of bonds between transforming DNA and the microprojectilefollowing delivery of the microprojectile to the target cell. This neednot be the case, however, as availability to a target cell may occur asa result of breakage of unbound segments of DNA or of other moleculesthat comprise the physical attachment to the microprojectile.Availability may further occur as a result of breakage of bonds betweenthe transforming DNA and other molecules, which are either directly orindirectly attached to the microprojectile. It further is contemplatedthat transformation of a target cell may occur by way of directrecombination between the transforming DNA and the genomic DNA of therecipient cell. Therefore, as used herein, a “coated” microprojectilewill be one that is capable of being used to transform a target cell, inthat the transforming DNA will be delivered to the target cell, yet willbe accessible to the target cell such that transformation may occur.

[0028] Any technique for coating microprojectiles that allows fordelivery of transforming DNA to the target cells may be used. Methodsfor coating microprojectiles that have been demonstrated to work wellwith the current invention have been specifically disclosed herein. DNAmay be bound to microprojectile particles using alternative techniques,however. For example, particles may be coated with streptavidin and DNAend-labeled with long chain thiol cleavable biotinylated nucleotidechains. The DNA adheres to the particles due to the streptavidin-biotininteraction but is released in the cell by reduction of the thiollinkage through reducing agents present in the cell.

[0029] Alternatively, particles may be prepared by functionalizing thesurface of a gold oxide particle, providing free amine groups. DNA,having a strong negative charge, binds to the functionalized particles.Furthermore, charged particles may be deposited in controlled arrays onthe surface of mylar flyer disks used in the PDS-1000 Biolistics device,thereby facilitating controlled distribution of particles delivered totarget tissue.

[0030] As disclosed above, it further is proposed that the concentrationof DNA used to coat microprojectiles may influence the recovery oftransformants containing a single copy of the transgene. For example, alower concentration of DNA may not necessarily change the efficiency ofthe transformation but may instead increase the proportion of singlecopy insertion events. In this regard, approximately 1 ng to 2000 ng oftransforming DNA may be used per each 1.8 mg of startingmicroprojectiles. In other embodiments of the invention, approximately2.5 ng to 1000 ng, 2.5 ng to 750 ng, 2.5 ng to 500 ng, 2.5 ng to 250 ng,2.5 ng to 100 ng, or 2.5 ng to 50 ng of transforming DNA may be used pereach 1.8 mg of starting microprojectiles.

[0031] For microprojectile bombardment transformation in accordance withthe current invention, both physical and biological parameters may beoptimized. Physical factors are those that involve manipulating theDNA/microprojectile precipitate or those that affect the flight andvelocity of either the macro- or microprojectiles. Biological factorsinclude all steps involved in manipulation of cells before andimmediately after bombardment, such as the osmotic adjustment of targetcells to help alleviate the trauma associated with bombardment, theorientation of an immature embryo or other target tissue relative to theparticle trajectory, and also the nature of the transforming DNA, suchas linearized DNA or intact supercoiled plasmids. It is believed thatpre-bombardment manipulations are especially important for successfultransformation of immature embryos.

[0032] Accordingly, it is contemplated that one may wish to adjustvarious of the bombardment parameters in small scale studies to fullyoptimize the conditions. One may particularly wish to adjust physicalparameters such as DNA concentration, gap distance, flight distance,tissue distance, and helium pressure. It further is contemplated thatthe grade of helium may affect transformation efficiency. One also mayoptimize the trauma reduction factors (TRFs) by modifying conditionsthat influence the physiological state of the recipient cells and thatmay therefore influence transformation and integration efficiencies. Forexample, the osmotic state, tissue hydration and the subculture stage orcell cycle of the recipient cells may be adjusted for optimumtransformation.

[0033] The present invention can be used with any transformable planttissue, including cells. By transformable as used herein is meant a cellor tissue that is capable of further propagation to give rise to aplant. Those of skill in the art recognize that a number of plant cellsor tissues are transformable in which after insertion of exogenous DNAand appropriate culture conditions the plant cells or tissues can forminto a differentiated plant. Tissue suitable for these purposes caninclude but is not limited to immature embryos, scutellar tissue,suspension cell cultures, immature inflorescence, shoot meristem, nodalexplants, callus tissue, hypocotyl tissue, cotyledons, roots, andleaves. Preferably, immature embryos are used as the explant of choice.

[0034] In an embodiment of the present invention, immature embryos (IEs)of corn are used as explants for Agrobacterium-mediated transformation.Corn ears are harvested approximately 6-16 days after pollination andused as a source of immature embryos. The present invention thusencompasses the use of freshly isolated embryos as described.

[0035] Any suitable plant culture medium can be used. Examples ofsuitable media would include but are not limited to MS-based media(Mursahige and Skoog, Physiol. Plant, 15:473-497, 1962) or N6-basedmedia(Chu et al., Scientia Sinica 18:659, 1975) supplemented withadditional plant growth regulators including but not limited to auxinssuch as picloram (4-amino-3,5,6-trichloropicolinic acid), 2,4-D(2,4-dichlorophenoxyacetic acid) and dicamba (3,6-dichloroanisic acid),cytokinins such as BAP (6-benzylaminopurine ) and kinetin, andgibberellins. Other media additives can include but are not limited toamino acids, macroelements, iron, microelements, vitamins and organics,carbohydrates, undefined media components such as casein hydrolysates,an appropriate gelling agent such as a form of agar, such as a lowmelting point agarose or Gelrite if desired. Those of skill in the artare familiar with the variety of tissue culture media, which whensupplemented appropriately, support plant tissue growth and developmentand are suitable for plant transformation and regeneration. These tissueculture media can either be purchased as a commercial preparation, orcustom prepared and modified. Examples of such media would include butare not limited to Murashige and Skoog (Mursahige and Skoog, Physiol.Plant, 15:473-497, 1962), N6 (Chu et al., Scientia Sinica 18:659, 1975),Linsmaier and Skoog (Linsmaier and Skoog, Physio. Plant., 18:100, 1965),Uchimiya and Murashige (Uchimiya and Murashige, Plant Physiol. 15:473,1962), Gamborg's B5 media (Gamborg et al., Exp. Cell Res., 50:151,1968), D medium (Duncan et al., Planta, 165:322-332, 1985), Mc-Cown'sWoody plant media (McCown and Lloyd, HortScience 6:453, 1981), Nitschand Nitsch (Nitsch and Nitsch, Science 163:85-87, 1969), and Schenk andHildebrandt (Schenk and Hildebrandt, Can. J. Bot. 50:199-204, 1972) orderivations of these media supplemented accordingly. Those of skill inthe art are aware that media and media supplements such as nutrients andgrowth regulators for use in transformation and regeneration and otherculture conditions such as light intensity during incubation, pH, andincubation temperatures that can be optimized for the particular varietyof interest.

[0036] Once the transformable plant tissue is isolated, the plant cellsin the tissue are transformed, and independently transformed plant cellsare selected. The independent transformants are referred to astransgenic events.

[0037] Those of skill in the art are aware of the typical steps in theplant Agrobacterium-mediated transformation process. The Agrobacteriumcan be prepared either by inoculating a liquid such as Luria Burtani(LB) media directly from a glycerol stock or streaking the Agrobacteriumonto a solidified media from a glycerol stock, allowing the bacteria togrow under the appropriate selective conditions, generally from about26° C.-30° C., or about 28° C., and taking a single colony or a smallloop of Agrobacterium from the plate and inoculating a liquid culturemedium containing the selective agents. Those of skill in the art arefamiliar with procedures for growth and suitable culture conditions forAgrobacterium as well as subsequent inoculation procedures. The densityof the Agrobacterium culture used for inoculation and the ratio ofAgrobacterium cells to explant can vary from one system to the next, andtherefore optimization of these parameters for any transformation methodis expected.

[0038] Typically, an Agrobacterium culture is inoculated from a streakedplate or glycerol stock and is grown overnight and the bacterial cellsare washed and resuspended in a culture medium suitable for inoculationof the explant. Suitable inoculation media for the present inventioninclude, but are not limited to ½ MS PL or ½MS VI (Table 1).

[0039] The next stage of the Agrobacterium mediated transformationprocess is the inoculation. In this stage the explants and Agrobacteriumcell suspensions are mixed together. In the present invention, immatureembryos are isolated directly into the inoculation medium containing theAgrobacterium. Embryos are cultured in inoculation media for less than30 min. The inoculation is generally performed at a temperature of about15° C. to 30° C., or about 23° C. to 28° C. The inoculation can also bedone by isolating the immature embryos directly onto the co-culturemedium (described below) and then spotting 1 μL of Agrobacteriumsolution onto the embryo or alternatively placing a piece of filterpaper saturated in Agrobacterium solution over the top of the embryosfor about 5 to 60 minutes. The filter paper and any excess solution arethen removed before co-culture.

[0040] After inoculation, any excess Agrobacterium suspension can beremoved and the Agrobacterium and target plant material are co-cultured.The co-culture refers to the time post-inoculation and prior to transferto a delay or selection medium. Any number of plant tissue culture mediacan be used for the co-culture step. For the present invention a reducedsalt media such as ½ MS-based co-culture media (Table 1) is used and themedia lacks complex media additives including but not limited toundefined additives such as casein hydolysate, and B5 vitamins andorganic additives. Plant tissues after inoculation with Agrobacteriumcan be cultured in a liquid media. More preferably, plant tissues afterinoculation with Agrobacterium are cultured on a semi-solid culturemedium solidified with a gelling agent such as agarose, or a low EEOagarose. The co-culture is typically performed for about one to threedays or for less than 24 hours at a temperature of about 18° C. to 30°C., or about 20° C. to 25° C. The co-culture can be performed in thelight or in light-limiting conditions. Usually, the co-culture isperformed in light-limiting conditions. By light-limiting conditions asused herein is meant any conditions that limit light during theco-culture period including but not limited to covering a culture dishcontaining the plant and Agrobacterium mixture with a cloth, foil, orplacing the culture dishes in a black bag, or placing the cultured cellsin a dark room. Lighting conditions can be optimized for each plantsystem as is known to those of skill in the art.

[0041] After co-culture with Agrobacterium or after bombardment with themicroprojectile, the explants can be placed directly onto selectivemedia. Previously, explants were subcultured onto selective media insuccessive steps or stages for 8 weeks or more. For example, the firstselective media could contain a low amount of selective agent, and thenext sub-culture could contain a higher concentration of selective agentor vice versa. The explants could also be placed directly on a fixedconcentration of selective agent and then subcultured repeatedly tomaximize selection. Alternatively, after co-culture with Agrobacteriumor immediately after bombardment, the explants could be placed on mediawithout the selective agent, or delay media, followed by selection asabove. All of these steps took a great deal of time, resulting inprolonged culture duration. In the present invention, it has beendiscovered that selection of transformed corn cells can take place muchfaster than was previously believed. Explants (regardless oftransformation method) are placed on selective media for from about 7 toabout 42 days, or from about 7 to about 30 days, or from about 7 toabout 21 days, or from about 7 to about 14 days. This limits the time inculture and eliminates several transfer steps, which makes the processmore efficient. As previously noted, further improvement in obtained byselecting under an elevated temperature. Thus, in at least oneembodiment of the invention, transformed corn explants are placed on aselection media at elevated temperature for a period of 2 weeks and thenmaintained for a further 1-2 weeks in the same selection media and inthe same vessel at a temperature of 27° C. or below.

[0042] Those of skill in the art are aware of the numerous modificationsin selective regimes, media, and growth conditions that can be varieddepending on the plant system and the selective agent. Typical selectiveagents include but are not limited to antibiotics such as geneticin(G418), kanamycin, paromomycin or other chemicals such as glyphosate orother herbicides. Additional appropriate media components can be addedto the selection or delay medium to inhibit Agrobacterium growth. Suchmedia components can include, but are not limited to, antibiotics suchas carbenicillin or cefotaxime. The cultures are subsequentlytransferred to a media suitable for the recovery of transformedplantlets. Those of skill in the art are aware of the number of methodsto recover transformed plants. A variety of media and transferrequirements can be implemented and optimized for each plant system forplant transformation and recovery of transgenic plants. Consequently,such media and culture conditions disclosed in the present invention canbe modified or substituted with nutritionally equivalent components, orsimilar processes for selection and recovery of transgenic events, andstill fall within the scope of the present invention.

[0043] To initiate a transformation process in accordance with thepresent invention, it is first necessary to select genetic components tobe inserted into the plant tissue. Genetic components can include anynucleic acid that is introduced into a plant tissue using the methodaccording to the invention. Genetic components can include non-plantDNA, plant DNA or synthetic DNA.

[0044] In a preferred embodiment, the genetic components areincorporated into a DNA composition such as a recombinant,double-stranded plasmid or vector molecule comprising at least one ormore of following types of genetic components: (a) a promoter thatfunctions in plant cells to cause the production of an RNA sequence, (b)a structural DNA sequence that causes the production of an RNA sequencethat encodes a product of agronomic utility, and (c) a 3′ non-translatedDNA sequence that functions in plant cells to cause the addition ofpolyadenylated nucleotides to the 3′ end of the RNA sequence.

[0045] The vector may contain a number of genetic components tofacilitate transformation of the plant tissue and regulate expression ofthe desired gene(s). In one preferred embodiment, the genetic componentsare oriented so as to express a mRNA, that in one embodiment can betranslated into a protein. The expression of a plant structural codingsequence (a gene, cDNA, synthetic DNA, or other DNA) that exists indouble-stranded form involves transcription of messenger RNA (mRNA) fromone strand of the DNA by RNA polymerase enzyme and subsequent processingof the mRNA primary transcript inside the nucleus. This processinginvolves a 3′ non-translated region that adds polyadenylated nucleotidesto the 3′ ends of the mRNA.

[0046] Means for preparing plasmids or vectors containing the desiredgenetic components are well known in the art. Vectors typically consistof a number of genetic components, including but not limited toregulatory elements such as promoters, leaders, introns, and terminatorsequences. Regulatory elements are also referred to as cis- ortrans-regulatory elements, depending on the proximity of the element tothe sequences or gene(s) they control.

[0047] Transcription of DNA into mRNA is regulated by a region of DNAusually referred to as the “promoter”. The promoter region contains asequence of bases that signals RNA polymerase to associate with the DNAand to initiate the transcription into mRNA using one of the DNA strandsas a template to make a corresponding complementary strand of RNA.

[0048] A number of promoters that are active in plant cells have beendescribed in the literature. Such promoters would include but are notlimited to the nopaline synthase (NOS) and octopine synthase (OCS)promoters that are carried on tumor-inducing plasmids of Agrobacteriumtumefaciens, the caulimovirus promoters such as the cauliflower mosaicvirus (CaMV) 19S and 35S promoters and the figwort mosaic virus (FMV)35S promoter, the enhanced CaMV35S promoter (e35S), the light-induciblepromoter from the small subunit of ribulose bisphosphate carboxylase(ssRUBISCO, a very abundant plant polypeptide). All of these promotershave been used to create various types of DNA constructs that have beenexpressed in plants.

[0049] Promoter hybrids can also be constructed to enhancetranscriptional activity, or to combine desired transcriptionalactivity, inducibility and tissue specificity or developmentalspecificity. Promoters that function in plants include but are notlimited to promoters that are inducible, viral, synthetic, constitutive,and temporally regulated, spatially regulated, and spatio-temporallyregulated. Other promoters that are tissue-enhanced, tissue-specific, ordevelopmentally regulated are also known in the art and envisioned tohave utility in the practice of this invention. As described below, itis preferred that the particular promoter selected should be capable ofcausing sufficient expression to result in the production of aneffective amount of the gene product of interest.

[0050] The promoters used in the DNA constructs (i.e.,chimeric/recombinant plant genes) of the present invention may bemodified, if desired, to affect their control characteristics. Promoterscan be derived by means of ligation with operator regions, random orcontrolled mutagenesis, etc. Furthermore, the promoters may be alteredto contain multiple “enhancer sequences” to assist in elevating geneexpression. Examples of such enhancer sequences have been reported byKay et al. (Science, 236:1299, 1987).

[0051] The mRNA produced by a DNA construct of the present invention mayalso contain a 5′ non-translated leader sequence. This sequence can bederived from the promoter selected to express the gene and can bespecifically modified so as to increase translation of the mRNA. The 5′non-translated regions can also be obtained from viral RNAs, fromsuitable eukaryotic genes, or from a synthetic gene sequence. Such“enhancer” sequences may be desirable to increase or alter thetranslational efficiency of the resultant mRNA. The present invention isnot limited to constructs wherein the non-translated region is derivedfrom both the 5′ non-translated sequence that accompanies the promotersequence. Rather, the non-translated leader sequence can be derived fromunrelated promoters or genes. Other genetic components that serve toenhance expression or affect transcription or translational of a geneare also envisioned as genetic components. The 3′ non-translated regionof the chimeric constructs should contain a transcriptional terminator,or an element having equivalent function, and a polyadenylation signalthat functions in plants to cause the addition ofpolyadenylatednucleotides to the 3′ end of the RNA. Examples of suitable3′ regions are (1) the 3′ transcribed, non-translated regions containingthe polyadenylation signal of Agrobacterium tumor-inducing (Ti) plasmidgenes, such as the nopaline synthase (NOS) gene, and (2) plant genessuch as the soybean storage protein genes and the small subunit of theribulose-1,5-bisphosphate carboxylase (ssRUBISCO) gene. An example of apreferred 3′ region is that from the ssRUBISCO E9 gene from pea(European Patent Application 385 962).

[0052] Typically, DNA sequences located a few hundred base pairsdownstream of the polyadenylation site serve to terminate transcription.The DNA sequences are referred to herein as transcription-terminationregions. The regions are required for efficient polyadenylation oftranscribed messenger RNA (mRNA) and are known as 3′ non-translatedregions. RNA polymerase transcribes a coding DNA sequence through a sitewhere polyadenylation occurs.

[0053] In one embodiment, the vector contains a selectable, screenable,or scoreable marker gene. These genetic components are also referred toherein as functional genetic components, as they produce a product thatserves a function in the identification of a transformed plant, or aproduct of agronomic utility. The DNA that serves as a selection devicefunctions in a regenerable plant tissue to produce a compound thatconfers upon the transformed plant tissue resistance to an otherwisetoxic compound when exposed to the compound in tissue culture media orin other media, including soil. Genes that may usefully be used as aselectable, screenable, or scorable marker include but are not limitedto GUS, green fluorescent protein (GFP), luciferase (LUX), antibioticslike kanamycin (Dekeyser et al., Plant Physiol., 90:217-223, 1989), andherbicides like glyphosate (EPSPS, Della-Cioppa et al., Bio/Technology,5:579-584, 1987), dalapon (deh), bromoxynil (bxn), sulfonyl herbicides(ALS, GST-II), bialaphos (bar), atrazine, (GST-III). Other selectiondevices can also be implemented including but not limited to toleranceto phosphinothricin, bialaphos, and positive selection mechanisms(Joersbo et al., Mol. Breed., 4:111-117, 1998)

[0054] The present invention can be used with any suitable planttransformation plasmid or vector containing a selectable or screenablemarker and associated regulatory elements as described, along with oneor more nucleic acids expressed in a manner sufficient to confer aparticular desirable trait. Examples of suitable structural genes ofagronomic interest envisioned by the present invention would include butare not limited to genes for insect or pest tolerance, herbicidetolerance, genes for quality improvements such as yield, nutritionalenhancements, environmental or stress tolerances, or any desirablechanges in plant physiology, growth, development, morphology or plantproduct(s).

[0055] Alternatively, the DNA coding sequences can effect thesephenotypes by encoding a non-translatable RNA molecule that causes thetargeted inhibition of expression of an endogenous gene, for example viaantisense- or cosuppression-mediated mechanisms (see, for example, Birdet al., Biotech Gen. Engin. Rev., 9:207-227, 1991). The RNA could alsobe a catalytic RNA molecule (i.e., a ribozyme) engineered to cleave adesired endogenous mRNA product (see for example, Gibson and Shillitoe,Mol. Biotech. 7:125-137, 1997). More particularly, for a description ofanti-sense regulation of gene expression in plant cells see U.S. Pat.No. 5,107,065 and for a description of RNAi gene suppression in plantsby transcription of a dsRNA see U.S. Pat. No. 6,506,559, U.S. PatentApplication Publication No. 2002/0168707 A1, and U.S. patentapplications Ser. No. 09/423,143 (see WO 98/53083), 09/127,735 (see WO99/53050) and 09/084,942 (see WO 99/61631), all of which areincorporated herein by reference. Thus, any gene that produces a proteinor mRNA that expresses a phenotype or morphology change of interest isuseful for the practice of the present invention.

[0056] Exemplary nucleic acids that may be introduced by the methodsencompassed by the present invention include, for example, DNA sequencesor genes from another species, or even genes or sequences that originatewith or are present in the same species, but are incorporated intorecipient cells by genetic engineering methods rather than classicalreproduction or breeding techniques. However, the term exogenous is alsointended to refer to genes that are not normally present in the cellbeing transformed, or perhaps simply not present in the form, structure,etc., as found in the transforming DNA segment or gene, or genes thatare normally present yet that one desires, e.g., to have over-expressed.Thus, the term “exogenous” gene or DNA is intended to refer to any geneor DNA segment that is introduced into a recipient cell, regardless ofwhether a similar gene may already be present in such a cell. The typeof DNA included in the exogenous DNA can include DNA that is alreadypresent in the plant cell, DNA from another plant, DNA from a differentorganism, or a DNA generated externally, such as a DNA sequencecontaining an antisense message of a gene, or a DNA sequence encoding asynthetic or modified version of a gene.

[0057] In light of this disclosure, numerous other possible selectableand/or screenable marker genes, regulatory elements, and other sequencesof interest will be apparent to those of skill in the art. Therefore,the foregoing discussion is intended to be exemplary rather thanexhaustive.

[0058] After the construction of the plant transformation vector orconstruct, said nucleic acid molecule, prepared as a DNA composition invitro

[0059] Those of skill in the art will appreciate the many advantages ofthe methods and compositions provided by the present invention. Thefollowing examples are included to demonstrate the preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments that are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

EXAMPLES Example 1

[0060] Bacterial Strains and Plasmids

[0061]Agrobacterium tumefaciens strain ABI is harbored with the binaryvectors pMON30113 (FIG. 1), pMON42071 (FIG. 2), or pMON65324 (FIG. 3).The T-DNA in pMON30113 contains a neomycin phosphotransferase II gene(nptII) as the selectable marker and a green fluorescence protein gene(gfp) screenable marker, both driven by 35S promoter, respectively.pMON42071 has 2 T-DNA, with nptII driven by an e35S promoterand GUSgenes by a rice actin promoter on one T-DNA and CP4 driven by a riceactin promoter and gfp genes by an e35S promoter on another. pMON65324has CP4 and gfp genes each driven by an e35S promoter also on the T-DNA.

Example 2

[0062] Preparation of Agrobacterium

[0063]Agrobacterium ABI containing a vector in glycerol stock isstreaked out on solid LB medium supplemented with antibiotics kanamycin(50 mg/L), spectinomycin (50 mg/L), streptomycin (50 mg/L) andchloramphenicol (25 mg/L) and incubated at 28° C. for 2 days. Two daysbefore Agrobacterium inoculation of the maize immature embryos, onecolony or a small loop of Agrobacterium from the Agrobacterium plate ispicked up and inoculated into 25 mL of liquid LB medium supplementedwith 100 mg/L of spectinomycin and 50 mg/L of kanamycin in a 250-mLflask. The flask is placed on a shaker at approximately 150 rpm and 26°C. overnight. The Agrobacterium culture is then diluted (1 to 5) in thesame liquid medium and put back on the shaker. Several hours later, oneday before inoculation, the Agrobacterium cells are spun down at 3500rpm for 15 min. The bacterium cell pellet is re-suspended in inductionbroth with 200 μM of acetosyringone and 50 mg/L spectinomycin and 25mg/L kanamycin and the cell density was adjusted to 0.2 at O.D.₆₆₀. Thebacterium cell culture (50 mL in each 250-mL flask) is then put back onthe shaker and grown overnight. On the morning of inoculation day, thebacterium cells are spun down and washed with liquid ½ MS VI medium(Table 1) supplemented with 200 μpM of acetosyringone. After one morespinning, the bacterium cell pellet is re-suspended in ½ MS PL medium(Table 1) with 200 μM of acetosyringone (Table 1) and the cell densitywas adjusted to 1.0 at O.D₆₆₀ for inoculation.

[0064] Reagents are commercially available and can be purchased from anumber of suppliers (see, for example Sigma Chemical Co., St. Louis,Mo.). TABLE 1 Media used in this invention¹. Co-culture InductionComponent ½ MSVI ½ MSPL medium MS MSW50 MS/6BA MSOD MS salts 68.5 g/l68.5 g/l 2.2 g/l 4.4 g/l 4.4 g/l 4.4 g/l 4.4 g/l Sucrose 20 g/l 68.6 g/l20 g/l 30 g/l 30 g/l 30 g/l — Maltose — — — — — — 20 g/l Glucose 10 g/l36 g/l 10 g/l — — — 10 g/l 1-Proline 115 mg/l 115 mg/l 115 mg/l 1.36 g/l1.38 g/l 1.36 g/l — Casamino Acids — — — 50 mg/l 500 mg/l 50 mg/l —Glycine 2 mg/l 2 mg/l 2 mg/l — 2 mg/l — — 1-Asparagine — — — — — — 150mg/l myo-Inositol 100 mg/l 100 mg/l 100 mg/l — 100 mg/l — 100 mg/lNicotinic Acid 0.5 mg/l 0.5 mg/l 0.5 mg/l 1.3 mg/l 0.5 mg/l 1.3 mg/l 1.3mg/l Pyridoxine · HCl 0.5 mg/l 0.5 mg/l 0.5 mg/l 0.25 mg/l 0.5 mg/l 0.25mg/l 0.25 mg/l Thiamine · HCl 0.1 mg/l 0.1 mg/l 0.6 mg/l 0.25 mg/l 0.6mg/l 0.25 mg/l 0.25 mg/l Ca Pantothenate — — — 0.25 mg/l — 0.25 mg/l0.25 mg/l 2,4-D — — 3 mg/l 0.5 mg/l 0.5 mg/l — — Picloram — — — 2.2 mg/l— — — Silver Nitrate — — 1.7 mg/l 1.7 mg/l — — — BAP — — — — — 3.5 mg/l—

Example 3

[0065] Explant Preparation

[0066] Several genotypes of corn were used in this study. Earscontaining immature embryos are harvested approximately 10 days afterpollination and kept refrigerated at 4° C. until use (up to 5 dayspost-harvest). The preferred embryo size for this method oftransformation is ˜1.0-2.0 mm. This size is usually achieved 10 daysafter pollination inside the green house with the growth conditions ofan average temperature of 87° F., day length of 14 hours withsupplemental lighting supplied by GE 1000 Watt High Pressure Sodiumlamps.

Example 4

[0067] Inoculation

[0068] Immature embryos are isolated from surface sterilized ears anddirectly dropped into the prepared Agrobacterium cell suspension in1.5-mL microcentrifuge tube. The isolation lasts continuously for 15min. The tube is then set aside for 5 min, which made the inoculationtime for individual embryos from 5 to 20 min. After Agrobacterium cellsuspension is removed using a fine tipped sterile transfer pipette, theimmature embryos are transferred onto the co-culture medium (Table 1).The embryos are placed on the medium with the scutellum side facing up.The embryos are cultured in a dark incubator (23° C.) for approximately24 h.

Example 5

[0069] Selection, regeneration and growth of transformants withparomomycin selection.

[0070] After the co-cultivation, the embryos were transferred onto amodified MS medium (Induction MS, Table 1) supplemented with 500 mg/Lcarbenicillin and 50, 100, 150 or 200 mg/L paromomycin in Petri dishes(100 mm×25 mm), 20 to 25 embryos per plate. The plates were kept in adark culture room at 27° C. for approximately 2 weeks. At the end of 2weeks, each piece of callus cultures from individual embryos wasexamined under a fluorescence stereomicroscope and the number of calluspieces with well-developed GFP-positive sectors were determined. All thecallus pieces were then transferred individually onto the firstregeneration medium, the same medium mentioned above except 2,4-D andpicloram were replaced by 3.5 mg/L BAP (MS/BAP, Table 1) and thecarbenicillin level was dropped to 250 mg/L. The cultures were moved toa culture room with 16-h light/8-h dark photoperiod and 27° C. After 5-7days, the callus pieces were transferred onto the second regenerationmedium, a hormone-free MS-based medium (MSOD, Table 1) in Petri dishes(100 mm×25 mm). In another 2 weeks, the callus pieces that had shootsregenerated or were still alive were transferred onto the samehormone-free medium in Phytatrays for further growth. Regenerated plants(R0) when they reached to the top of Phytatrays and had one or morehealthy roots were moved to soil in peat pots in a growth chamber. In 7to 10 days, they were transplanted into 12-in pots and moved togreenhouse with conditions for normal corn plant growth. The plants wereeither self-pollinated or crossed with wild-type plants.

[0071] Seed Set and Transgene Segregation in R1 Generation:

[0072] Immature ears from some of the R0 plants were harvested atdifferent times after pollination. The immature embryos from all or someof the kernels were isolated and cultured on MSOD medium supplementedwith 200 mg/L paromomycin. The embryos were examined under thefluorescence microscope to determine the number of embryos with GFPexpression. In approximately 7 to 10 days, the number of embryosgerminated to seedlings was also determined. Other plants were grown tomaturity and seeds were harvested from individual plants.

[0073] Development of Transformants:

[0074] Almost all the embryos inoculated and co-cultured withAgrobacterium for one day showed excellent GFP gene transientexpression, which was demonstrated by a great number of single-celledGFP-positive spots on each embryo. The embryos were transferred afterone day coculture onto the selective callus induction medium, a modifiedMS medium (Induction MS, Table 1) supplemented with 500 mg/Lcarbenicillin and 50, 100, 150 or 200 mg/L paromomycin. The cultureswere examined periodically for the development of GFP-positive sectors.It was observed that most of the GFP-positive spots resulting from thetransient expression disappeared within 3-4 days. On the fourth or fifthday, small multiple-celled GFP-positive spots started to emerge on someof the embryos. Those spots gradually developed into GFP-positivesectors that were easily identified under the fluorescence microscope.Some of the embryos could form more than one GFP-positive sector. In twoweeks, one fifth to one third of the callus pieces (one callus piecefrom one embryo) in different experiments showed well-developedGFP-positive sectors. Once the callus pieces were transferred onto theregeneration medium, some of the GFP-positive sectors started toregenerate into shoots. Usually roots would not develop until they hadbeen on the regeneration medium for at least 3 weeks, that is when theywere already in Phytatrays. Some of the plantlets were big enough to bemoved to soil after growing in Phytatrays for 2 to 3 weeks (7 to 8 weeksafter inoculation). It might take another 2 to 3 weeks to move all theplantlets to soil. Before the plantlets were planted in soil, they wereexamined under the fluorescence microscope again. Based on all thetransgenic events moved to soil in five experiments, approximately 48%of the events had one GFP-positive plant, 52% had 2 to 8 plants.

[0075] Transformation Frequency:

[0076] One fifth to one third of inoculated embryos developed to calluspieces with GFP-positive sector(s) after being cultured on the callusinduction medium with paromomycin. However, it appeared that majority ofthem were not able to regenerate to plants. Some of them regeneratedinto small shoots on the regeneration medium but did not grow further.That could be caused by low expression of the transgene or other unknownreasons. At the end, 2 to 5% of transformation efficiencies wereachieved based on the number events with GFP-positive plants moved tosoil.

[0077] Elimination of Non-transformed Escapes:

[0078] In our experiments, it only took 7 to 10 weeks from inoculationto R0 transgenic plants moved to soil. The reduction of overallselection time could make it easier for nontransformed tissue to surviveand eventually regenerate to plants if selection stringency is notproperly modified, which was reflected in our early experiments. Forexample, in two experiments, 100 mg/L paromomycin was used at each stageof selection. The level of selection was apparently not high enough,because only 41 and 33%, respectively, of the plants survived theselection and moved to soil were GFP-positive. This high level of escaperate was not acceptable practically and also suggested that theselection stringency should be adjusted. Two approaches were tested toeliminate the non-transformants: 1) increased paromomycin level at eachstage, but kept the same; 2) kept the paromomycin at 100 mg/L in earlystages, but increased it to 200 mg/L at the last stage of selection (inPhytatrays). To test the first approach, three levels of paromomycin(100, 150 and 200 mg/L) were compared. The results showed in Table 2suggested that as the paromomycin level in medium increased from 100 to150 to 200 mg/L, the proportion of GFP-positive plants also increasedfrom 67% at 100 mg/L level to 75% at 150 mg/L and to 83% at 200 mg/L. InExperiment 3370, all the tissue were selected on medium containing 100mg/L paromomycin, but the paromomycin level was increased to 200 mg/L atthe last stage of selection (in Phytatrays). Among the plants moved tosoil in this experiment 72% were GFP-positive, which was much higherthan in Experiments 3307 and 3324. These results suggest that the escaperate could be significantly reduced by increasing the selectionstringency starting from beginning or at the last stage of selection,although more study is necessary to find an optimal selection regime.TABLE 2 Effect of selection stringency on escape rate. # plants to soilExp-Treat # Selection regime^(a) Total GFP+ GFP− 3307 P100 61 25 (41%)36 (59%) 3324 P100 45 15 (33%) 30 (67%) 3368-1 P100 6  4 (67%)  2 (33%)3368-2 P150 12  9 (75%)  3 (25%) 3368-3 P200 30 25 (83%)  5 (17%) 3370P100 → P200 25 18 (72%)  7 (28%)

[0079] Using this system, transformed R0 plants were transferred to soilapproximately two months after the explants were inoculated withAgrobacterium. Because the amount of tissue culture manipulation wassignificantly reduced, the time of production of high quality transgenicmaize plants was reduced, and maize transformation was much lesstedious. It should also work for other explant materials besidesimmature embryos.

[0080] Example 6

[0081] Selection, Regeneration and Growth of Transformants Through NPTIISelection

[0082] Immature embryos from a different corn line were inoculated andco-cultivated with Agrobacterium ABI::pMON30113 as described earlier.The embryos were then transferred onto a modified MS medium (MSW50,Table 1) supplemented with 100 or 200 mg/L paromomycin and 500 mg/Lcarbenicillin to inhibit Agrobacterium in Petri dishes (100 mm×25 mm).The cultures were incubated in a dark culture room at 27° C. for 2-3weeks. All the callus pieces were then transferred individually onto thefirst regeneration medium (MS/6BA, Table 1) supplemented with the samelevels of paromomycin. The cultures were grown on this medium and in aculture room with 16-h light/8-h dark photoperiod and 27° C. for 5-7days. They were then transferred onto the second regeneration medium(MSOD, Table 1) in Petri dishes (100 mm×25 mm) for approximately 2weeks. All the callus pieces with regenerating shoots and living tissuewere transferred onto the same medium contained in Phytatrays for shootsto grow further before being moved to soil. It took 2-4 weeks. Theregeneration media (MS6BA and MSOD) were all supplemented with 250 mg/Lcarbenicillin and 100 or 200 mg/L paromomycin.

[0083] Transformation Frequency Through NPTII Selection:

[0084] In 2 to 3 weeks, a number of callus pieces developed sectorsexpressing GFP. In two experiments, the number of callus piecesdeveloped GFP-expressing sectors varied significantly. It was close to24% in one experiment, and 61.5% in another. Most of the sectors wereapparently non-regenerable and only a small number of them regeneratedinto small shoots. Eventually, similar to the first corn line, 2.4 and3.3% of transformation efficiency were achieved in these twoexperiments, respectively.

EXAMPLE 7

[0085] Selection, Regeneration and Growth of Transformants ThroughGlyphosate Selection

[0086] Embryos inoculated and co-cultured with AgrobacteriumABI::pMON42071 or pMON65324 were selected on the callus induction medium(MSW50, Table 1) supplemented with 0.1 or 0.25 mM glyphosate and 500mg/L carbenicillin for approximately 3 weeks. All the callus piecesdeveloped from individual embryos were regenerated the same way andunder the same conditions as described in last section for the NPTIIselection, except the MS6BA and MSOD media were supplemented with 250mg/L carbenicillin and 0.1 or 0.25 mM glyphosate instead of paromomycin.

[0087] Transformation Frequency Through Glyphosate Selection:

[0088] Five experiments were conducted with either ABI::pMON42071 orABI::pMON65324. Inoculated immature embryos were selected on the callusinduction medium, MSW50 supplemented with 0.1 or 0.25 mM glyphosate forapproximately 3 weeks. Almost all the embryos were able to develop somecallus tissue, and they were all moved onto the regeneration mediumsupplemented with 0.1 or 0.25 mM glyphosate for plant regeneration andfurther selection. The transformation efficiency varied from experimentto experiment with a range from 0.5 to 3.1%. Unlike in the experimentswith NPTII selection, all regenerated plants from the glyphosateselection experiments were GFP-positive.

Example 8

[0089] Selection Temperature

[0090] This example describes the use of increased incubationtemperature during the selection process. The usual temperature is 27°C. Immature embryos were transformed as described above and selectedwith glyphosate as in Example 7, with the exception of the Induction MScontaining 4.4 g/L MS salts, 30 g/L sucrose, 1.38 g/L proline, 500 mg/Lcasamino acids, 100 mg/L inositol, 0.5 mg/L nicotinic acid, 0.5 mg/Lpyridoxine, 0.6 mg/L thiamine, 0.5 mg/L 2,4-D, 1.7 mg/L silver nitrateand 20 mg/L BAP. During the first two weeks of selection, temperaturesof 27° C., 30° C., 32° C. and 34° C. were compared. After two weeks, thetemperature was reduced to 27° C. for the final week of selection. Therewas also the addition of a rooting media (MSOD with 0.75 mg/L IBA and0.5 mg/L NAA and 0.1 mM glyphosate) during the final regeneration step.With an average of 5 experiments, 27° C. gave a transformation frequencyof 3.6%, 30° C. was 6.7%, 32° C. was 6.9%, and 34° C. was 8.4% (Table3). The higher temperatures of 32° C. and 34° C. gave an increasednumber of abnormal albino or striped events. The earlier transformationfrequencies are for normal events. Experiments were also done thatcompared 27° C. with 30° C. A direct comparison of the two temperaturesduring the same experiment had a transformation frequency of 3.4% for27° C. and 9.9% for 30° C. Over a large number of transformations at 27°C., transformation frequency averaged 5.5%. At 30° C., transformationfrequency averaged 15%. Overall, the increase in temperature duringselection increased the transformation frequency by about twofold. TABLE3 Effect of Callus Induction Temp. on TF of the CP4 Agro-mediated TfnSystem # Normal # Albino or Trt. Events to Striped TF % TF % Exp. # Temp# Explants Soil Events (all events) (normal events) 4915 27 C. 261 19 17.7 7.3 30 C. 261 29 1 11.5 11.1 32 C. 261 22 0 6.4 8.4 34 C. 259 16 27.7 6.9 4921 27 C. 350 0 0 0.0 0.0 30 C. 346 16 1 5.5 5.2 32 C. 337 16 15.6 5.3 34 C. 344 19 1 5.6 5.5 4922 27 C. 220 7 0 3.2 3.2 30 C. 220 6 02.7 2.7 32 C. 220 6 1 3.2 2.7 34 C. 220 7 2 4.1 3.2 4929 27 C. 369 13 03.5 3.5 30 C. 369 29 4 6.9 7.9 32 C. 369 24 2 7.0 6.5 34 C. 369 47 514.1 12.7 4931 27 C. 344 16 1 4.9 4.7 30 C. 347 26 1 7.6 7.5 32 C. 34236 2 11.1 10.5 34 C. 342 36 3 12.0 11.1 Total 27 C. 1544 55 2 3.7 3.6 30C. 1545 106 7 7.4 6.7 32 C. 1529 106 6 7.3 6.9 34 C. 1534 129 13 9.3 8.4

Example 9

[0091] Detection of GFP Expression

[0092] Inoculated immature embryos, callus tissue developed from theembryos, and regenerated shoots were examined periodically under a LeicaMZ80 dissecting microscope equipped with a filter set (HQ480/40excitation, HQ535/50 emission and Q505LP dichroic mirror), especially atone day after inoculation and the end of each selection and regenerationphases. All the R0 plants were also examined for the GFP expressionbefore being moved to soil.

[0093] Seed Set and Transgene Segregation in R1 Generation:

[0094] Immature ears of some of the R0 plants from NPTII selection wereharvested at different times after pollination. The immature embryosfrom all or some of the kernels were isolated and cultured on MSODmedium supplemented with 200 mg/L paromomycin. The embryos were examinedunder the fluorescence microscope to determine the number of embryoswith GFP expression. In approximately 7 to 10 days, the number ofembryos germinated to seedlings was also determined.

1. A method for producing a transformed maize plant comprising the stepsof: inserting into a transformable maize tissue a nucleic acidcomprising a selectable marker gene to obtain a transformed maizetissue; culturing the transformed maize tissue for a period of time fromabout 7 days to about 42 days at a temperature of from about 28° C. toabout 35° C. in a selection media containing a selection compound thatinhibits the growth of non-transformed maize tissue and permits thecontinued growth of transformed maize tissue; identifying and selectingtransformed maize tissue that grows in the selection media; andregenerating a transformed maize plant from the selected transformedmaize tissue.
 2. The method of claim 1 wherein the period of time in theselection media is between about 7 days and about 28 days.
 3. The methodof claim 1 wherein the selection temperature is from about 30° C. toabout 34° C.
 4. The method of claim 3 wherein the selection temperatureis 30° C.
 5. The method of claim 3 wherein the selection temperature ismaintained for a period of about 1-14 days.
 6. The method of claim 1wherein the selection is performed in a single vessel without replacingor replenishing the selection media during the selection period.
 7. Themethod of claim 1 wherein the selection compound is a herbicide.
 8. Themethod of claim 7 wherein the herbicide is selected from the groupconsisting of glyphosate, bialophos, phosphinothricin or Basta.
 9. Themethod of claim 1 wherein the nucleic acid is inserted into the maizetissue by inoculation with an Agrobacterium containing said nucleicacid.
 10. The method of claim 9 wherein the Agrobacterium inoculation isperformed for less than about 20 minutes.
 11. The method of claim 9wherein the Agrobacterium inoculation is performed by contacting thetransformable maize tissue with filter paper saturated with theAgrobacterium containing the nucleic acid.
 12. The method of claim 11wherein the filter paper contacts the transformable maize tissue forbetween about 5 and about 60 minutes.
 13. The method of claim 9 where inthe Agrobacterium inoculation is performed by spotting the maize tissuewith about 1 μL of Agrobacterium containing the nucleic acid.
 14. Atransgenic maize plant produced by the method of claim
 1. 15. A methodfor producing a transformed cereal plant comprising the steps of:inserting into a transformable cereal tissue a nucleic acid comprising aselectable marker gene to obtain a transformed cereal tissue; culturingthe transformed cereal tissue for a period of time from about 7 days toabout 42 days at a temperature of from about 28° C. to about 35° C. in aselection media containing a selection compound that inhibits the growthof non-transformed cereal tissue and permits the continued growth oftransformed cereal tissue; identifying and selecting transformed cerealtissue that grows in the selection media; and regenerating a transformedcereal plant from the selected transformed cereal tissue.
 16. The methodof claim 15 wherein the period of time in the selection media is betweenabout 7 days and about 28 days.
 17. The method of claim 15 wherein theselection temperature is from about 30° C. to about 34° C.
 18. Themethod of claim 17 wherein the selection temperature is 30° C.
 19. Themethod of claim 17 wherein the selection temperature is maintained for aperiod of about 1-14 days.
 20. The method of claim 15 wherein theselection is performed in a single vessel without replacing orreplenishing the selection media during the selection period.
 21. Themethod of claim 15 wherein the selection compound is a herbicide. 22.The method of claim 21 wherein the herbicide is selected from the groupconsisting of glyphosate, bialophos, phosphinothricin or Basta.
 23. Themethod of claim 15 wherein the nucleic acid is inserted into the cerealtissue by inoculation with an Agrobacterium containing said nucleicacid.
 24. The method of claim 23 wherein the Agrobacterium inoculationis performed for less than about 20 minutes.
 25. The method of claim 23wherein the Agrobacterium inoculation is performed by contacting thetransformable cereal tissue with filter paper saturated with theAgrobacterium containing the nucleic acid.
 26. The method of claim 25wherein the filter paper contacts the transformable maize tissue forbetween about 5 and about 60 minutes.
 27. The method of claim 9 where inthe Agrobacterium inoculation is performed by spotting the maize tissuewith about 1 μL of Agrobacterium containing the nucleic acid.
 28. Atransgenic cereal plant produced by the method of claim
 15. 29. A methodfor increasing the transformation efficiency of a cereal transformationprocess comprising limiting the anaerobiosis effect during theinoculation of Agrobacterium to the transformable cereal tissue.